Device and methods for identifying and treating aspirin non-responsive patients

ABSTRACT

The present invention relates to methods and compositions for identifying and treating subjects in need of antithrombotic therapies but who are not responsive to aspirin.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application No.60/636,744, filed Dec. 14, 2004, which is herein incorporated byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Aspirin Effects and Aspirin Resistance

Aspirin is known to reduce post-acute myocardial infarction (AMI)cardiovascular events by 25-30%. Recent findings indicate that theincidence of aspirin non-responders is between 5-40% of the treatedpopulation. It has been proposed that this sub-population of patientsdoes not benefit from aspirin treatment and has an increased risk forfuture cardiovascular events. Although bedside devices exist formonitoring platelet function, these devices were not designed tospecifically examine aspirin non-responsiveness. Therefore, the realprevalence of aspirin non-responders remains unknown. Moreover, existingbedside platelet monitoring devices are affected by platelet P2 receptorP2Y₁₂ antagonism. P2Y₁₂ antagonism induced by Plavix which is commonlyprescribed for patients with acute coronary syndromes (ACS). Since thispatient population is at high risk for thrombotic events, and has beenfound to have a high incidence of aspirin non-responsiveness, and sincePlavix requires aspirin co-therapy to be maximally effective, effectivemanagement of this patient population requires that new plateletmonitoring devices and methods be employed.

One method for detecting aspirin resistance in the background of Plavixuse comprises the step of evaluating simultaneously in real time theeffect of aspirin on thrombus formation triggered by a collagen-coatedcapillary and by arachidonic acid-induced whole blood thrombus formationunder arterial shear rates.

The antithrombotic properties of aspirin were reported more than 50years ago and are mostly attributed to the inhibition of prostaglandinsynthesis. Aspirin treatment is known to exhibit both anti-thromboticand anti-inflammatory activities, thought to be mediated by irreversibleacetylation of Ser530 of cycloxygenase type 1 (Cox-1) and Ser516 ofcycloxygenase type 2 (Cox-2), respectively. Cox-1 is constitutivelyexpressed whereas Cox-2 is inducible. This is of particular importancewhen evaluating the anti- and potential pro-thrombotic activities ofaspirin. As platelets are anucleate cells, aspirin inhibition of thesynthesis of the pro-aggregatory and vasoconstrictor metabolitethromboxane A2 (TxA2) lasts for the life of the cell even thoughaspirin's half life is only 20 minutes. By comparison, aspirininhibition of Cox-2, and the subsequent prevention of prostacyclin(PGI2) by endothelial cells is rapidly overcome by newly synthesizedCox-2. Resynthesis of Cox-2 is judged to be beneficial as PGI2 isantiaggregatory and vasodilatory. Benefits of aspirin therapy incardio/cerebrovascular diseases are well documented. For example,aspirin is known to reduce the incidence of AMI in the acute phase ofunstable angina by up to 25% (Lancet, 2(8607):349-60 (1988); Bmj,308(6921):81-106 (1994)), and to reduce mortality and recurrent strokein patients with acute ischemic stroke (Lancet, 349(9065):1569-81(1997)). A meta-analysis recently suggested that the use of aspirin inpatients population with diabetes and peripheral arterial disease beexpanded (Bmj 324(7329):71-86 (2002)).

However, in the past 15 years there have been several studies reportingthe existence of a sub-population of patients resistant to theantithrombotic activity of aspirin. The prevalence of aspirin resistanceis reported to range between 5 and 40%. The broad range is attributed tothe absence of a reliable assay that will specifically assess theantithrombotic properties of aspirin.

Several hypotheses have been proposed for aspirin resistance, some ofwhich concern aspirin, others not: Cox-2 expression in platelets, Cox-2activation in inflammatory and vascular cells, and/or production ofeicosanoids, increased reaction to collagen or adenosine diphosphate(ADP), presence of erythrocytes, interaction between aspirin andnon-steroidal anti-inflammatory drugs (NSAIDs), variant isoforms ofCox-1, increased platelet turnover, poor compliance, increased amount ofplasma von Willebrand factor (vWF), or genetic polymorphisms (ofglycoprotein IIb-IIIa (GP IIb-IIIa), GP Ia-IIa, and eventually GP Ibα).Several studies have shown that aspirin resistance may be inducible. Forexample, one study showed that about 30% of people become resistantwhile under chronic aspirin therapy (Helgason, C. M. et al., Stroke,25(12):2331-6 (1994)) and another showed that an increase in percentageof aspirin-resistant patients occurs following surgical procedures (i.e.post coronary artery bypass grafting (CABG), Zimmermann, N. et al., JThorac Cardiovasc Surg, 121(5):982-4 (2001)). Others have reported aprogressive reduction in platelet sensitivity to aspirin as measured byplatelet aggregation in long-term treated patients (Pulcinelli, F. M. etal., J Am Coll Cardiol, 43(6):979-84 (2004)). Moreover, the lack ofreliable tools that specifically measure aspirin effects may directlycontribute to the extent of the aspirin resistance phenomenon. Indeed,aspirin resistance may be also dose-dependent, and it is plausible thatpart of the aspirin resistant population could benefit from apersonalized therapy. The development of an assay that will allowquantitative assessment of these issues is therefore necessary.

The clinical consequences of aspirin resistance are of major importanceas it is now commonly accepted that it correlates with futurecardiovascular events (Gum, P. A. et al., J Am Coll Cardiol, 41(6):961-5(2003); Eikelboom, J. W. et al., Circulation, 105(14):1650-5 (2002)). Anexample of aspirin resistance that may correlate with thrombotic eventsis the interaction of aspirin with NSAIDs. It has been reported that theaspirin-dependent inhibition of platelet aggregation and serum TxB2formation (stable metabolite of TxA2) was compromised when ibuprofen wasadministered prior to aspirin (Catella-Lawson, F. et al., N Engl J Med,345(25):1809-17 (2001)). The binding of ibuprofen is proposed to blockthe S530 site of Cox-1 before its irreversible acetylation by aspirin.This important finding appears to correlate with clinical events as arecent study has highlighted an increased cardiovascular mortality inpatients combining ibuprofen plus aspirin vs aspirin alone (MacDonald,T. M. et al., Lancet, 361(9357):573-4 (2003)). The importance of aspirinresistance is further emphasized by the state of the art combinationaspirin/P2Y₁₂ inhibition (Plavix), demonstrated to confer higherantithrombotic efficacy than single therapies alone.

A clear, unequivocal definition of aspirin resistance becomes crucial asit could ultimately lead to a personalized antithrombotic strategy. Theneed for individualized screening is reinforced by the fact aspirincauses gastro-intestinal (GI) and bleeding complications. The GItoxicity of aspirin appears to be dose-dependent, starting with doses aslow as 10 mg/day. Since other platelet-inhibiting and anticoagulantagents potentiate the gastro-intestinal lesions and bleeding riskassociated with low-dose aspirin, one can therefore question the use ofaspirin in aspirin-resistant patients. Moreover, aspirin's side effectsare not limited to GI and bleeding complications. Aspirin is thought toinduce asthma in as much as 20% of the patients (Jenkins, C. J. et al.,Bmj, 328(7437):434 (2004)). This situation is rendered even moreparadoxical as a high percentage of patients with aspirin-induced asthmaalso take NSAIDs (notably ibuprofen).

Laboratory Methods to Detect Aspirin Resistance:

Several laboratory tests of platelet function have been designed and areavailable to “diagnose” aspirin-resistance using whole blood. Mostcertainly, the 2 main tools utilized are the Ultegra Rapid PlateletFunction Assay (RPFA-ASA), and the PFA-100 device.

The RPFA-ASA cartridge has been specifically designed to address thelevel of inhibition of platelet aggregation achieved by aspirintreatment. As mentioned by the manufacturer, it is a qualitative measureof the effects of aspirin. In that assay, fibrinogen-coated beadsagglutinate platelets through binding to GP IIb-IIIa receptors followingstimulation by metallic cations and propyl gallate. The change inoptical signal triggered by the agglutination (light transmittanceincreases as activated platelets bind and agglutinate the beads in thewhole blood suspension) is measured. A recent study has detected a highincidence (23%) of aspirin non-responsiveness using this device, anddetermined a history of coronary artery disease to be associated withtwice the odds of being an aspirin non-responder (Wang, J. C. et al., AmJ Cardiol, 92(12):1492-4 (2003)). Aspirin resistance cannot be evaluatedby the RPFA assay, however, in patients who were prescribed either GPIIb-IIIa inhibitors, dipyridamole, plavix (or ticlid), or NSAIDS(ibuprofen, naproxen, diclofenac, indomethacin, piroxicam) since thosecompounds interfere with the assay.

In the PFA-100 device, the platelet hemostatic capacity (PHC) of acitrated blood sample is determined by the time required for a plateletplug to occlude a 150 μM aperture cut into a collagen-epinephrine coatedmembrane (used for the detection of aspirin). In the PFA-100 system,samples of citrated blood are aspirated through the aperture at shearrates of ˜4,000-5,000/sec. Under these high conditions of shear, vWFinteractions with both GP Ibα and GP IIb-IIIa trigger the thromboticprocess. In the context of clinical events, plasma levels of vWF areexpected to increase following platelet-rich thrombi formation andendothelial cell injury. Interestingly, Chakroun et al. reported thatthe aspirin-resistant population measured by PFA-100 also demonstratedan increased plasma vWF ristocetin cofactor activity (Chakroun, T. etal., Br J Haematol, 124(1):80-5 (2004)). Furthermore, a poor inhibitionof thrombotic events has been reported for shear rates around 10,000/secwith aspirin (Barstad, R. M. et al., Thromb Haemost, 75(5):827-32(1996)), and PFA-100 detects desmopressin (DDAVP) therapy whichincreases plasma levels of vWF (Fressinaud, E. et al., Br J Haematol,106(3):777-83 (1999)). One can therefore argue that the high incidenceof aspirin resistance found post-AMI with the PFA-100 device (Gum, P. A.et al., Am J Cardiol, 88(3):230-5 (2001)) reflects an increase inplatelet sensitivity towards high shear induced collagen-vWF/GP Ibα-/GPIIb-IIIa-interactions (Chakroun, T. et al., Br J Haematol, 124(1):80-5(2004)) rather than true aspirin resistance. In addition, severalinvestigators have found that most of the aspirin-resistant populationidentified by PFA-100 appeared to be aspirin sensitive as shown byinhibition of platelet aggregation induced by arachidonic acid (AA)(Gum, P. A. et al., J Am Coll Cardiol, 41(6):961-5 (2003); Chakroun, T.et al., Br J Haematol, 124(1):80-5 (2004)), as well as extremely lowTxB₂ levels (Andersen, K. Thromb Res, 108(1):37-42 (2002)). Moreover,investigators have reported a good prognosis for long term clinicalevents in the aspirin-resistant population revealed by AA-inducedplatelet aggregation, but this was not the case for the PFA-100 device.The data described herein also show that the PFA-100 device does notspecifically reveal aspirin effects.

Other models have been utilized for examining aspirin effects, such asplatelet aggregation and bleeding time. For example, arachidonicacid-induced platelet aggregation in platelet rich plasma is classicallyconsidered the gold standard for evaluation of aspirin effects onplatelets. Nevertheless, determining aspirin resistance should beinvestigated using whole blood for several reasons. First, erythocytesmay contribute to aspirin-resistance. Valles et al. (Valles, J. et al.,Blood, 78(1):154-62 (1991)) have demonstrated that the presence oferythrocytes directly affects platelet reactivity by increasing TxB₂synthesis, release of serotonin (5-HT), β-thromboglobulin (β-TG) andADP. This has a major impact on the evaluation of aspirin resistance, asthe same authors found that aspirin treatment (200-300 mg daily) failedto block platelet reactivity in presence of erythrocytes in two thirdsof a group of patients with ischemic heart disease and ischemic stroke,despite full inhibition of TxA2 synthesis (Valles, J. et al.,Circulation, 97(4):350-5 (1998)).

Second, nucleated cells (leukocytes) may contribute toaspirin-resistance. It is commonly accepted that because platelets areanucleated cells, their potential for producing TxA₂ is irreversiblysuppressed during their lifetime after aspirin treatment. However, apotential source of aspirin-insensitive TxA₂ and 8-epi-prostaglandin-F2α (8-epi PGF_(2α)) (2 known platelet agonists) exists as the de novosynthesis of Cox-2 and full recovery of Cox activity occurs 2 to 4 hoursafter aspirin treatment via stimulated nucleated cells (Maclouf, J. G.et al., Thromb Haemost, 79(4):691-705 (1998)). Platelet microparticlescan also indirectly participate in generation of Cox-2-dependent TxA₂formation via delivery of AA by sPLA₂. Similarly, it has been reportedthat AA derived from neutrophils can increase TxA₂ formation inplatelets (Maugeri, N. et al., Blood, 80(2):447-51 (1992)).

Third, plasma proteins and lipids may contribute to aspirin-resistance.The presence of F2-isoprostanes in plasma are increased in patients withunstable angina, diabetes mellitus, hypercholesterolemia and cigarettesmokers. These sub-populations present some of the highest percentage ofaspirin-resistant patients. One candidate for mediating aspirinresistance is the 8-epi-PGF_(2α) or isoprostane 8-epi prostaglandinF_(2α) (8-iso-PGF₂α). It is produced from AA by nonenzymatic lipidperoxidation catalyzed by oxygen free radicals, and isaspirin-insensitive (Wang, Z. et al., J Pharmacol Exp Ther,275(1):94-100 (1995)). Consequently, 8-epi-PGF_(2α) generation can occurin all cellular players of atherosclerosis. It is a potentvasoconstrictor and has been reported to activate platelets at highconcentrations and cause dose-dependent irreversible plateletaggregation in the presence of subluminal concentrations of collagen,ADP or arachidonic acid. In addition, 8-epi-PGF_(2α) could potentiatethe expression of GP IIb-IIIa and enhance platelet adhesion onfibrinogen. Although a direct interaction of 8-epi-PGF_(2α) with the TPαreceptor has been shown, the biological significance of 8-epi-PGF_(2α)in thrombosis remains controversial as low concentrations (such as thosefound in vivo) may inhibit collagen-induced platelet aggregation butamplify low dose ADP-induced platelet aggregation (Yin, K. et al., JPharmacol Exp Ther, 270(3):1192-6 (1994)).

Whole blood platelet aggregation and whole blood aggregation utilizing ascreen filtration pressure method have been shown to detect the effectsof aspirin. However, these are labor intensive techniques and theevaluation of the aggregation process is performed under low shear rateconditions (like those encountered in veins) which do not reflect thethrombotic process occurring in moderately stenosed arteries.

Template bleeding times have been demonstrated to be accurate indetermining platelet function prior to and following ASA administration.This technique is sensitive enough to diagnose platelet dysfunction(notably von Willebrand Disease), but bleeding time is highly variable,and sensitive to all anti-platelet agents, anti-coagulants, and otherfactors such as alcohol and green tea.

Altogether, the different techniques are either time-consuming, or ofpoor prognostic for true aspirin resistance. This demonstrates the needfor a new device and methods that will specifically evaluate aspirineffects.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method of evaluatingaspirin responsiveness in a subject, comprising:

(a) perfusing a blood sample from a subject through a channel in aperfusion device, wherein the channel has a coating which produces ablood deposit when exposed to blood and wherein the perfusion devicecomprises a pump which draws the blood through the channel at a selectedshear rate, producing a blood deposit; and

(b) determining a status of aspirin responsiveness by measuring thelevel of blood deposited in the chamber and comparing the measured levelof deposited blood with a control level of deposited blood, wherein saidcontrol level is selected from the group consisting of (1) a levelmeasured using a sample of blood from said subject which has beentreated with aspirin; (2) a level measured using a sample of blood fromsaid subject which has been contacted with a thromboxane A2 receptorantagonist; and (3) a level previously determined to correspond to astatus of aspirin responsiveness.

In another embodiment, the present invention provides a method ofqualifying a subject for treatment with a thromboxane A2 receptorantagonist, comprising:

(a) perfusing a blood sample from said subject through a channel in aperfusion device, wherein the channel has a coating which produces ablood deposit when exposed to blood and the perfusion device comprises apump coupled to the outlet end of the housing to draw the blood throughthe channel at a desired shear rate, producing a blood deposit, andwherein said blood sample is treated with an amount of a platelet ADPreceptor antagonist sufficient to inhibit thrombosis at leastapproximately 20% relative to an untreated sample; and

(b) wherein said blood sample is treated with an amount of aspirinsufficient to cause at least an approximately 50% inhibition ofthrombosis in a blood sample relative to an untreated sample; and

(c) wherein a subject is qualified for treatment with a thromboxane A2receptor antagonist if less than approximately 50% inhibition ofthrombosis is observed in said blood sample relative to an untreatedsample.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and constitutepart of this specification, illustrate an embodiment of the invention,and, together with the general description given above and the detaileddescription given below, serve to explain the features of the invention.

FIG. 1: FIG. 1 shows that the effects of Plavix will prevent the use ofthe RPFA-ASA cartridge in the ACS patient population. Whole blood wasdrawn into 3.2% Sodium citrate vacuum tubes and analyzed using theUltegra RPFA-ASA assay. Sample results are based on the extent ofplatelet aggregation, reported in Aspirin Reaction Units (ARU). ARU≧250is consistent with no platelet dysfunction, ARU<550 is consistent withplatelet dysfunction due to aspirin.

FIG. 2A is a schematic view of a perfusion device used in theaggregation of platelets to image and analyze thrombus formations.

FIG. 2B is a flowchart of an embodiment of operation of the perfusiondevice of FIG. 2A.

FIG. 2C is an alternative depiction of a perfusion device of FIG. 2A.

FIG. 2D is a schematic of another perfusion device used in theaggregation of platelets to produce thrombus formations and also used inthe imaging and analysis of the formations.

FIG. 2E is a flowchart of the operation of the perfusion device of FIG.2D.

FIG. 2F is an illustration of the perfusion device of FIG. 2D.

FIG. 2G is a preferred embodiment of a socket used in the perfusiondevice of FIGS. 2A and 2D.

FIG. 2H is a series of still images of thrombus formations produced bythe perfusion device of FIG. 2A.

FIG. 3A-C are cross-sectional views of various examples of a member usedin the perfusion device of FIG. 2 to aggregate platelets and producethrombus formations.

FIG. 4A-4D are views of another example of the member.

FIG. 4E-4G are a top and plan views of another example of the member.

FIG. 4H are top and plan views of another example of the member in FIGS.4E-G.

FIG. 4I-4K are plan and perspective views of another example of themember.

FIG. 4L-4M are perspective views of another example of the member inFIGS. 4I-4K.

FIG. 5 is a schematic view of an a control system for use with theinstruments of FIGS. 2A and 2D;

FIG. 6 shows the effects of aspirin and 50% inhibition of P2Y₁₂ onarachidonic acid-induced platelet aggregation in PRP, TxB2 generationusing PFA-100 with both Collagen/Epinephrine (CEpi) and Collagen/ADP(CADP) cartridges. Whole blood was drawn into 3.8% Sodium citrate vacuumtubes and analyzed using the Platelet Function Analyzer (PFA-100).Sample results are based on the platelet function under high shearstress. Platelet adhesion and aggregation are triggered by abiologically active membrane coated with collagen and either epinephrine(EPI) or ADP. Platelets exposed to EPI or ADP under flow conditions areactivated on the collagen surface. The time required to full occlusionof an aperture cut in the membrane defines the “closure time” (CT).

FIG. 7 shows a thrombotic profile in the perfusion chamber. Meanthrombus volumes quantified on semi-thin cross sections were plottedagainst their corresponding mean grey levels using Prism Software. Thetop of the figure corresponds to cross sections performed inside the400×250 μm rectangle that was analyzed for the mean grey level at 5 mmfrom the proximal part of the capillary. Capillary chambers wereperfused with either PPACK-anticoagulated blood treated with increasingdoses of Eptifibatide (Integrilin) at 750/sec (empty squares), or withnon-anticoagulated blood at 1500/sec (black squares). Bar=120 μm. Doublebar=100 μm. The sole measurement of the grey level is now performed todetermine mean thrombus volume.

FIG. 8A shows representative pictures of the thrombotic process formingin the rectangular perfusion chamber at different time points.Phe-Pro-Arg-chloromethylketone (PPACK)-anticoagulated blood is added toa tube containing Rhodamine 6-glucose (Rhodamine 6G) (0.2 mg/ml). Fiveminutes later, blood is perfused through a rectangular capillary mountedon the stage of an inverted microscope (under ultraviolet (UV) light(Rhodamine filter) platelets appear fluorescent). As the blood flows onthe collagen type III-coated capillary chamber, platelets adhere andstart building a thrombus. Two frames per second are recorded andanalyzed for mean fluorescent intensity for the entire perfusion period.At the end of the perfusion, standard deviation in light intensity isplotted against the time by the Prism software. FIG. 8B represents thethrombotic profiles of untreated or Integrilin-treated blood (flatcurve).

FIG. 9 is a representation of the thrombotic deposits formed on type IIIcollagen at 1000/sec in the rectangular perfusion chamber in 2 blooddonors, before and after aspirin uptake (325 mg/d for 3 days).Anticoagulated blood (citrate 3.8%, 1:9 v of blood) is collected andadded into 5 ml tubes containing either DMSO (vehicle control for theP2Y₁₂ antagonist), a direct P2Y₁₂ antagonist at 5 μM (which totallyblock the receptor), or a direct P2Y₁₂ antagonist at 5 μM in presence of20 μM Epinephrine. Anticoagulated blood is perfused through arectangular capillary (0.2×2 mm, Vitrocom) coated with fibrillar typeIII collagen at 1000/sec for 4 minutes. Thrombotic deposits are thenrinsed for 15 seconds with buffer “C”, fixed for 1 minute inphosphate-buffered glutaraldehyde (2.5%), stained for 45 sec intoluidine blue. Picture is taken at 5 mm from the proximal part of thechamber, and measurement of the grey level performed with Prismsoftware. Note that aspirin effect is observed on dimethylsulfoxide(DMSO)-treated blood and P2Y₁₂ antagonism+epinephrine-treated blood.

FIG. 10 is a schematic representation of the AA/shear-induced thromboticprocess utilized in our preliminary study. AA (0.8 mM) is added tocitrate-anticoagulated blood (37° C. incubation time=1 minute) thenperfused via a silastic tubing to a stenosis (high shear rate, 1700/sec:Hγ) followed by a low shear rate area (80/sec: Lγy). Observation of themixed platelet/leukocyte aggregates is performed under UV at 10×magnification using a fluorescent microscope.

FIG. 11 shows how TP antagonism induces dethrombosis.

FIG. 12A shows aspirin background and FIG. 12B shows how the aspirinbackground blocks dethrombosis activity of TP antagonist.

FIG. 13 shows how PGD2 induces dethrombosis on aspirin background.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor methods, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. Further, unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention pertains. In describing and claiming thepresent invention, the following terminology and grammatical variantswill be used in accordance with the definitions set forth below.

The term “perfusing” means moving a fluid through, over or acrosssomething such a particular media.

Perfusion Assays

In one embodiment, the present invention provides a method of evaluatingaspirin responsiveness in a subject, comprising perfusing a blood samplefrom a subject through a channel in a perfusion device. Perfusiondevices have existed for over 30 years and were designed in order tocharacterize the thrombotic process under shear conditions. Severaldifferent types of perfusion chambers are described in the literatureand can be classified according to their geometry (circular, annular,flat chambers) or the surfaces (blood vessels, isolated proteins)exposed to the blood flow. Examples of perfusion devices are disclosedin U.S. Patent Application No. 60/635,659, filed Dec. 14, 2004, and U.S.patent application entitled “A Device for Aggregating, Imaging andAnalyzing Thrombi and a Method of Use”, filed on Dec. 14, 2005 havingMorgan, Lewis & Bockius LLP Attorney Docket No. 044481-5091-PR which areherein incorporated by reference in their entirety.

One example of a perfusion device is shown in FIG. 2A. FIG. 2A is aschematic diagram of a perfusion device 10, in the form of a kineticaggregometer instrument for capturing the kinetics or a moving ortime-lapse image of thrombus formation, coagulation, leukocyte or tumorcell recruitment in a blood sample. To image the thrombus formation, theperfusion device 10 may use microscopy and/or micro-videographytechniques, and in other embodiments light microscopy techniques. Shownin FIG. 2B is a flowchart of the operation of the perfusion device 10.Referring to both FIGS. 2A & 2B, the perfusion device 10 includes amember 12, a fluid handling assembly 14, an imaging assembly 15, and adata analyzer 16. According to box steps 2 and 3 in FIG. 2B, a sample ofblood can be pre-treated with an imaging agent or fluorescent label andmoved or perfused through member 12 by the fluid handling assembly 14for a period of time so as to initiate thrombus formation within themember 12. Alternatively, the imaging agent can be added to the sampleduring the perfusion process. The imaging assembly 15 in box step 5repeatedly images the developing thrombus formation within the member 12during the perfusion using a camera 124 capable of motion capture. Theimaging assembly 15 can use light microscopy and/or micro-videographytechniques with fluorescence illumination. The image can be captured astime-lapsed digital image data and integrated over time to provide amovie or motion picture display of the evolving thrombus formation as isindicated by step boxes 6 and 7. In addition, the digital image data canbe processed and correlated by analyzer 16 to quantify a temporalevolution of volume of thrombus formation or other quantifiablecharacteristics of thrombi formation, as is indicated by step boxes 6and 8. This information can be useful in determining the real timeefficacy of a given anti-thrombotic therapy using, for example: aspirin,P2Y₁₂ receptor targeted compounds and GPIIb/IIIa antagonists, Integrilinas well as other platelet-thrombus modulators, and can serve as feedbackinformation to modifying the dosage of the therapy. The imaging assembly15 can additionally include a non-imaging photodetector 127 thatgenerates a signal in response to the fluorescence intensity of thethrombus formation. The signal can be used by the data analyzer 16 tocorrelate and quantify, in an alternate manner, the temporal evolutionof the thrombus volume, in addition to other quantifiablecharacteristics of thrombus formation.

Alternatively as shown in FIG. 2D the perfusion device 10′ can beconfigured for fixed imaging or “end-point measurement” of thrombi.Specifically, perfusion device 10′ is configured for imaging thethrombus formation at a fixed point in time, preferably at theconclusion of the thrombus formation process using light microscopytechniques. Shown in FIG. 2E is a flow chart of the operation theperfusion device 10′ in FIG. 2D.

Perfusion device 10′, like perfusion device 10 of FIG. 2A, can alsogenerally include a member 12, a fluid handling assembly 14, an imagingassembly 15, and an analyzer 16. Referring to both FIGS. 2D and 2E, thefluid handling assembly 14 of perfusion device 10′ perfuses or moves asample of blood through member 12 for a period of time so as to initiatethrombus formation within the member 12. The sample of blood can besubsequently treated with image enhancing agents that fix and stain thethrombus formation within the member 12, as is shown by step boxes 2 aand 2 b. The image enhancing agents can be delivered by the fluidhandling assembly 14. The imaging assembly 15 can image the thrombusformation formed within the member 12 using microscopy techniques knownto one of ordinary skill in the art, as indicated in step boxes 4 and 5.The imaging assembly 15 of perfusion device 10′ may use light microscopywith Köhler illumination. The imaging assembly 15 can additionallycapture the image as digital image data using a camera 124. The digitalimage data can be further processed by analyzer 16 in order to determinethe volume of thrombus formation and other quantifiable characteristicsof thrombus formation, such as for example, height, area and perimeterof the thrombus formation.

The member 12 can be configured for capturing the thrombus formation tobe imaged and may be used in systems using either kinetic imaging orfixed end-point imaging of the thrombus formation. The member 12, shownfor example in FIG. 2A, can be configured such that the surfaces of themember 12 define a flow channel 18 having an inlet end 20 and an outletend 22. At least one of the surfaces 26 defining the channel 18 istransparent so as to make the blood sample in the flow channel visiblefor purposes of observing the thrombus formation under known microscopyor micro-videography techniques.

The transparent surface 26 is typically made of a non-thrombogenicmaterial, for example, silica materials such as quartz, fused silica,boro silicate glass, plexi-glass or any other glass or plastic surfaceappropriate for thrombus formation when coated and capable of imagingformation readouts. Member 12 can be made completely of transparentnon-thrombogenic material, such as where member 12 is, for example, amicro-capillary tube having a substantially circular cross-section 24.For example, member 12 may be a micro-capillary tube with a centralthrough bore defining flow channel 18. As seen in FIG. 2A, the flowchannel 18 defines a longitudinal axis A-A along which the sample ofblood can flow. Typically, flow channel further defines a holding volumeof about 201 μl or less, although channel 18 can be configured to holdlarger volumes to suit a given assay. Referring to FIGS. 3A-3C, the flowchannel 18 further defines a cross-sectional area 24 perpendicular tothe longitudinal axis A-A which can be of any geometry. Thecross-sectional area 24 may be substantially rectangular in shape asseen in FIG. 3A, or alternatively the cross-sectional area 24 can besubstantially circular in shape, as is shown in FIG. 3B or substantiallysemi-circular in shape, as shown in FIG. 3C, although otherconfigurations are possible.

The flow channels 18 of FIGS. 3A-3C define a channel width “d” andheight “h”. Preferably, height h is about 200 microns and width d ofabout 2 mm, more preferably less than about 1.5 mm, even more preferablyless than about 1 mm, even more preferably less than about 500 micronsand yet even more preferably less than about 400 microns. The channelwidth d can be constant along longitudinal axis A-A, or alternativelythe width d can vary along the longitudinal axis. Varying the width d offlow channel 18 changes the shear rate characteristics of the bloodmoving through the member 12. This permits a single member 12 to be usedto study thrombus formations under varying shear rates of blood flow.

Coatings which Produce a Blood Deposit

Within the method of the present invention, the channel has a coatingwhich produces a blood deposit when exposed to blood. At least one ofthe surfaces defining the channel 18 can include a coating 25 at aconcentration so as to facilitate thrombus formation in the channel 18.The coating 25 can coat all the surfaces of member 12 defining channel18, for example, as seen in FIGS. 3A and 3B or alternatively less thanall the surfaces may be coated, for example, as seen in FIG. 3C.Preferably, the transparent surface 26 is provided with the coating 25.Blood flowing through channel 18 comes in contact with and reacts withthe coating 25 thereby initiating thrombus formation within the flowchannel 18. The coating may be a thrombogenic material like a collagen,for example, fibrillar collagen type III or fibrillar collagen type I oralternatively, fibrinogen or a tissue factor (for example, thromborel),although any desired platelet agonists, vascular adhesive proteins,pro-coagulant, pro-inflammatory material, adhesive matrix orchemoattractant may be used. The concentration of coating 25 can dependon the material used or the extent of thrombus formation sought. Forexample, collagen can be used at a concentration of about 10 μg percentimeter-squared. In addition, different coatings 25 may used incombination in a single member 12 to test anti-thrombotic efficacy undervarying conditions. For example, fibrillar collagen type III or I can beused to evaluate the anti-platelet agents directed against GP Ib/IX/V,collagen receptor, GPIIb/IIIa, the ADP receptor in combination withaspirin and hirudin. In another example, fibrinogen can provideinformation about the GPIIb/IIIa pathway and level of inhibition. In yetanother example, thromborel can be used to evaluate anti-thromboticactivity of thrombin receptor antagonists. Alternatively, selectins maybe used in place of or along with the coatings 25 to attract circulatingcells, such as leukocytes. Alternatively, fibronectin with chemokinesmay be used to attract circulating cells. To test the anti-thrombotictherapy using different thrombotic agonists, member 12 can be configuredto include multiple channels 18 that can run substantially parallel toaxis A-A. Thus in one embodiment, the coating which produces a blooddeposit is selected from a group consisting of fibrillar collagen typeIII, fibrillar collagen type I, fibrinogen and tissue factor. In anotherembodiment, said coating is human type III collagen especially whereinsaid subject has been administered aspirin, further comprising theaddition of an ADP-receptor antagonist to the sample, wherein saidconcentration of said antagonist in the sample is sufficient to cause a50% inhibition of platelet aggregation relative to a sample which hasbeen treated with aspirin but not treated with said ADP-receptorantagonist.

Substantially Flat Transparent Housing

In one embodiment, said method uses a perfusion device furthercomprising a substantially flat transparent housing, wherein the channelextends through the housing with an inlet end for receiving a continuousflow of blood and an outlet end for discharging the blood. Shown inFIGS. 4I-4M is yet another example of member 12 in the form of aperfusion chamber member 12″. Perfusion chamber member 12″, shown inperspective view in FIGS. 4J and 4K shows an example of a substantiallyflat housing 54. Housing 54 can be formed of two mating portions: upperhousing 56 and lower housing 58. Lower and upper housing 56, 58 portionsmay be joined so as to form a fluid tight seal therebetween, for exampleby heat sealing, joint adhesive sealing or any other techniques known toone of ordinary skill in the art for fluid tight sealing.

Lower housing 58 can be a substantially flat, housing having body 68defining a flow channel system 18′ substantially along longitudinal axisA-A through which a blood sample can be moved. In one example, channelsystem 18′ includes a single inlet channel 40 which splits into twosubstantially parallel flow channels 70, 72 which terminate respectivelyat outlets 50, 52 coterminous with the body 68. Alternatively, flowchannels 70, 72 can be configured with independent inlets. Flow channels40, 70, and 72 define cross-sectional area 24 which may be circular,although other cross-sectional geometries are possible. Moreover, thecross-sectional geometry can vary along the longitudinal axis, forexample transitioning from substantially rectangular to substantiallycircular along the longitudinal axis or vice versa. Flow channels 40, 70and 72 each define a diameter d′ which may vary along the channel 18′ inthe direction of axis A-A. Alternatively, diameter d′ may be constantalong the axis A-A. In addition, the dimensions or geometry of thecross-sectional area 24 of flow channels 70 can be different than thecross-sectional area of flow channel 72. Flow channels 70, 72 can beconfigured such that their total holding volume may be smaller thanabout 20 μl, although larger holding volumes can be provided for a givenapplication. Upper housing 56 can be a substantially flat plate definingan interior surface 62 in communication with the channel system 18′.

Coating 25, as previously described, may be coated along a portion ofthe interior surface 62 for facilitating thrombus formation in thechannel system 18′ when the blood sample is moved therethrough. Morespecifically, the coatings 25 are applied along a portion interiorsurface 62 in communication with channels 70, 72 to facilitate thrombusformation therein. The coatings 25 used in, for example, flow channel 70can be different than the coating 25 used in flow channel 72 to observevarying anti-thrombotic reactions. For example, the coating 25 in flowchannel 70 may be of a different type than the coating 25 in flowchannel 72, or alternatively, the coating 25 in channel 70 may vary inconcentration from the coating used in channel 72. Upper housing 56 maybe made from a transparent non-coating in order to facilitate themicro-videography or microscopy imaging of the thrombus formations inflow channels 70, 72.

The member 12″ shown in FIG. 4K includes two substantially parallel flowchannels 70 and 72. In an alternative example, as shown in FIGS. 4L and4M, the perfusion member 12′″ can include at least three flow channels82, 84 and 86. Each flow channel 82, 84 and 86 can be separatelyconfigured in a manner similarly described with respect to flow channels70 and 72. In addition, each channel 82, 84, and 86 can have a surface80, 90, 92 in communication with the channel 82, 84, and 86 that iscoated with varying coatings 25. Alternatively, member 12′″ may beconfigured so as to define as many flow channels in the system ofchannels 18″ as is needed for a blood therapy study.

Referring back to FIGS. 2C and 2F, perfusion device 10,10′ can include areceiver member or socket 38 configured for holding and orienting member12 in a specific manner with respect to the remaining components ofperfusion device 10. More specifically, socket 38 can be configured soas to properly secure and orient member 12 for proper imaging of thethrombus formations within channel 18. Socket 38 can be a holder 39including a chamber 37 for housing the member 12 and tubing. Forexample, shown in FIG. 2G is another example of a holder 39 having achamber 37 for housing the member 12. Socket 38 can be furtherconfigured to hold piping, for example, a single silastic tubing from ablood sample reservoir to the member 12 and another silastic tubing fromthe member 12 to the pump (not shown).

In another example, socket 38 can have a connection fitting thatcomplementarily mates with the connection fitting of micro-capillarytube member 12 such that the transparent surface 26 is oriented withrespect to imaging assembly 15 in order to image the thrombus formationinside channel 18 with the appropriate resolution and magnification. Forexample, socket 38 can include a telescopic stage that could be operatedto bring the channel 18 into focus with respect to imaging assembly 15.

Socket 38 can be further configured so as to properly secure and orientmember 12 for a liquid tight connection to the blood sample source,imaging agent source and fluid handling assembly 14. For example, socket38 can include fluid handling fittings and elements known to one ofordinary skill in the art so as to, for example, properly deliver ablood sample or imaging agent flow channel 18. More specifically, socket38 can include, for example, a quick disconnect coupling to permit easyand quick insertion and disconnection of member 12 from a fluid handlingelement of the fluid handling assembly 14, for example, a pump. Inanother example where member 12 can be embodied as a microchip member12, perfusion device 10 can include a socket 38 for complimentary“snap-in” arrangement with microchip member 12, thus facilitating easychange-out of the microchip member 12 and set up of perfusion device 10for multiple assays.

Pump and Fluid-Handling

Within the method of the present invention, the perfusion devicecomprises a pump which draws the blood through the channel. Referringagain to the schematics of FIGS. 2A and 2D, perfusion device 10, 10′includes fluid handling assembly 14 which can have one portion 14 a forhandling delivery of a blood sample to member 12 and moving the bloodsample through the channel 18. Fluid handling assembly 14 can haveanother portion 14 b for handling delivery of other liquids, (not shownin FIG. 2A) for example, image enhancing agents to channel 18.

Fluid handling portion 14 a can move a blood sample through channel 18of member 12 by vacuum pressure. As seen in FIGS. 2C and 2D, fluidhandling portion 14 a can be single tubing, for example silastic tubingconnected to inlet and outlet ends 20, 22 of member 12 to connect to thereservoir sample of blood and the syringe pump. For example, and as seenin FIG. 41, flow channels 70 and 72 can be connected at their outletends 50, 52 to separate syringes 104 a, 104 b respectively. Syringes 104a, 104 b can be conventional type syringes including pistons forcreating a vacuum. Syringes 104 a, 104 b can be connected to a pump 106to operate the pistons of syringes 104 a, 104 b. Pump 106 can be acommercially available peristaltic pump, for example, a HarvardApparatus Pump. Additionally, fluid handling portion 14 b can includetubing, valves and connection fittings to draw blood from a samplesource and deposit the sample to a waste vessel upon exit from member12. Preferably, all tubing, connections and fluid handling elements aremade of non-thrombogenic material.

Fluid handling portion 14 b can be configured to deliver various imagingenhancing agents to facilitate proper imaging of the thrombus formation.For example, in kinematic imaging of the thrombus formation in channel12, preferably a fluorescent label, for example, Rhodamine 6G in saline,is added directly to the sample of blood so as to reach a concentrationof about 1-20 micrograms/ml. Alternatively, the blood can be fluorescedusing Mepacrine at a concentration of about 0.2 mg/ml as a dye. The dyecan be added to the whole sample prior to or during perfusion. Inaddition, a blood sample to be kinematically imaged may be slightlyanti-coagulated. The fluid handling assembly 14 can be configured todeliver a small amount of anti-coagulant, for example, PPACK, citrate,heparin, EDTA, a factor Xa inhibitor or any other anti-coagulant knownin the art, to the blood sample prior to perfusion. Alternatively, thecoating can be fluorescently labeled. Quenching of the fluorescentsurface due to platelet deposition or an other cells becomes theread-out of the thromobotic process.

Fluid handling portion 14 b can be configured for facilitating fixed endpoint measurement imaging or other alternative imaging techniques tomicro-videography. For example, after fluid handling portion 14 a movesor perfuses a blood sample through channel 18 so as to initiate thrombusformation, fluid handling portion 14 b can deliver image enhancingagents to fix and stain the thrombus formation within the channel 18 inaccordance with, for example, light microscopy techniques know to one ofordinary skill in the art. Imaging enhancing agents can include: (i) arinsing buffer; (ii) a fixing solution of either PBS or glutaraldehyde2.5% or PBS, PFA 4%; and (iii) a stain solution, i.e. toluidin bluesolution form Becton Microscopy Science. Fluid handling assembly 14 caninclude the requisite tubing, piping and handling elements needed fordelivery of the image enhancing agents to the channel 18. In addition, acontrol system can be interfaced with fluid handling portion 14 b toautomate the sequencing and metering control of the delivery of theimage enhancing agents.

Fluid handling assembly 14 can include one or more fluid controlelements 100, for example, a valve that controls the flow of the bloodsample into the blood sample channel 18. Any piping components, fittingand/or elements located between the blood sample reservoir and thetubing member 12 may be constructed from non-thrombogenic material andpreferably constructed so as not to disturb the laminar flow of theblood sample through member 12 in order to avoid activating theplatelets. These fluid control elements 100 can be configured forautomatic operation by a properly interfaced control system.

In the case of where member 12 is specifically embodied as the microchipmember 12 of FIGS. 4E and 4H described above, the microchip member 12can include fluid handling portion 14 b that delivers the imageenhancing agents, i.e. dye, fixing agent, rinsing buffer, etc. Morespecifically, microchip member 12 can include liquid ports 30, 32, and34 of fluid handling assembly 14. Each of liquid ports 30, 32 and 34 canbe configured for delivery of any one of the image enhancing agents. Theliquid ports 30, 32 and 34 can be configured so as to deliver the imageenhancing agents directly into the channel 18. Alternatively, themicrochip member 12 can include only a single liquid port, for example,liquid port 30 to deliver all the necessary image enhancing agents.

Shear Rates

Within the method of the present invention, the pump draws the bloodthrough the channel at a selected shear rate, producing a blood deposit.A blood sample can be moved through channel 18 of member 12 at a userselected shear rate which is expressed in units of blood per second(s−1). For example, the blood sample can be moved through channel 18 ata shear rate that mimics the human arterial shear rate estimated to beabout 600 to about 800 units per second, shear rates found in moderatestenosed arteries of about 1,500 to about 10,000 units per second oralternatively mimic the human venous shear rate of about 50 to about 200units per second. In this manner, a blood assay using perfusion device10 can model thrombus formation in a vein or artery. In addition, theshear rate of flow through member 12 can be selected so as to accountfor stenosis, where a moderately stenosed artery can result in a shearrate of about 1,500 units per second, and a severely stenosed artery canresult in a shear rate of about 6000 units per second.

Shear rate can be a function of both the volumetric flow rate “Q” andthe cross-sectional geometry of the channel through which a fluid flows.For example, where channel 18 defines a substantially rectangularcross-sectional area 24 having a width “a” and a height “b,” the shearrate at the wall shown in equation (1):γ_(at wall)=1.03*Q/(a*b ²)  (1)

Where cross-sectional area 24 is substantially circular having a radius“r” the shear rate is found by the equation (2):γ_(at wall)=4*Q/(π*r ³)  (2)

In order to regulate or adjust the shear rate to mimic blood flowthrough veins or arteries, the flow rate can be adjusted by accordinglychanging the flow rate of the pump or otherwise changing the geometry ofthe channel 18. For example, as previously described, member 12 can beconfigured so as to vary the width d of channel 18 in the direction offlow along the longitudinal axis A-A. In one embodiment, the selectedshear rate is an arterial shear rate.

Measuring the Level of Blood Deposited in the Channel

Within the method of the present invention, the status of aspirinresponsiveness is determined by measuring the level of blood depositedin the channel and comparing the measured level of deposited blood witha control level of deposited blood. In one embodiment, the measurementof the rate of blood deposit comprises obtaining images of the depositedblood, and calculating the levels of deposited blood from the images,wherein said images are obtained at least once every ten seconds duringperfusion.

One method of obtaining images is through use of an imaging assembly.One example of an imaging assembly is shown in FIGS. 2A and 2B. Imagingassembly 15 may be configured for kinematic imaging of the thrombusformation or recruitment of any circulating blood cells in channel 18 ofmember 12 using light microscopy and/or micro-videography techniquesinvolving fluorescence illumination as is known in the art. Imagingassembly 15 of perfusion device 10 includes fluorescence excitationoptics, to imaging a time-lapse video or motion picture of thrombusformation. Referring to FIGS. 2A and 2B, imaging assembly 15 ofperfusion device 10 includes fluorescence excitation optics, forexample, a light source 122 and a microscope 120 interfaced with acamera 124 for imaging a time-lapse video or movie of thrombusformation. Camera 124 may be a CCD camera with microscopic zoomcapability to eliminate the need for a separate microscope. Camera 124can be, for example, a Nikon DXM1200 digital camera. Camera 124 may be adigital monochrome video camera having 8-bit, integration times ca. 500ms, IEEE1394 interface wherein images are acquired at 1-3 Hz. Microscope120 may have for example a magnification of 20× and includes excitationand emission filters and a dichroic mirror. Light source 122 may be anLED, and more preferably, light source 122 can be a high power green LEDhaving a preferred wavelength of about 530 nm with a narrow spectraldistribution and low power consumption. Alternatively, multiplefluorescent measurements, for example using red or blue LED can beenabled to perform complex assays in which a computer controlledanalyzer can support the wavelength, exposure and flow parameters of theexperiment including saving the data for analysis.

Shown in FIG. 2C is an arrangement of perfusion device 10 showingrelative positions of the member 12, fluid handling assembly 14, andimaging assembly 15 in an enclosure 17. The imaging assembly 15 isdisposed proximate the member 12. Specifically, member 12, light source122 and the objective of microscope 120 can be disposed relative to oneanother such that the light source 122 can illuminate the channel 18 andthe microscope 120 can magnify and resolve the thrombus formation inchannel 18 as the thrombus formation develops. The microscope 120 can bedisposed relative to the transparent surface 26 of member 12 in order tofocus on the thrombus formation in channel 18. The enclosure 17 isconfigured to substantially house the perfusion device 10 and alsofilter or block out surrounding room lighting so as not to interferewith the fluorescence imaging of the thrombus formation.

During perfusion of the fluorescent labeled blood sample through member12, the blood sample reacts with the coating 25 to begin thrombusformation within channel 18. Fluorescent platelets adhere to the coatedsurface, thus initiating aggregation of individual platelets to form thethrombi. The imaging assembly 15 repeatedly images the thrombusformation developing in channel 18. The thrombus formation adheres andaggregates along the surfaces of channel 18 coated with coating 25. Thefluorescent labeled platelets appear in the field of view of themicroscope 120. The illumination from the light source 122 passingthrough member 12 visually enhances the view of the fluoresced thrombusformation. The lenses of the microscope 120 resolve and magnify theimage of the thrombus formation with sufficient contrast so as to enableimage capture and analysis of the formation.

The camera 124 of imaging assembly 15 captures the fluoresced image ofthe evolving thrombus formation as digital image data, a sample of whichis shown in FIG. 2H. The frame rate of the camera 124 of imagingassembly 15 may be about 2 frames per second to capture the thrombusformation as a time-lapse motion picture. Other frame rates are possiblebut may require larger image data file sizes and hardware. The digitaldata image can be stored to read/write digital medium 137 in, forexample, a hard drive of a computer or alternatively a networked datastorage device.

Imaging assembly 15 can alternatively and optionally include anon-imaging photodetector 127, for example, a photodiode orphotomultiplier. The photodetector 127 produces an electrical signalresponse to light emitted from the fluoresced thrombus formation. Theelectrical signal can be read, processed, and correlated by computer 136to quantify the temporal evolution of thrombus formation and any othercharacteristics of the thrombus formation. The photodetector 127 can beused to provide a more sensitive, better signal to noise measurement ofthrombus formation in parallel with the time-lapse video.

In addition, perfusion device 10 can be configured for performing bothkinematic time lapse imaging of the thrombus formation and alternatefixed end point measurement imaging. In order to perform fixed end pointmeasurement imaging, perfusion device 10 can be configured in a manneras described below with respect to perfusion device 10′.

Alternatively, imaging assembly 15 can be configured for fixed end pointimaging of the thrombus formation in channel 18 of member 12 using lightmicroscopy techniques and optics involving Köhler illumination as isknown in the art. In contrast to the kinetic imaging of thrombusformation, fixed end point imaging captures a point in time image, the“end point” of the thrombus formation after perfusion of the bloodsample through the member 12 and after the thrombus formation has beenfixed and stained in the channel 18. Shown in FIG. 2D, is a schematicview of perfusion device 10′ and imaging assembly 15 relative to themember 12. Preferably, imaging assembly 15 includes a light microscope120 and a light source 122. Light source 122 may be an LED for example ahigh power green LED.

Shown in FIG. 1F is an arrangement of perfusion device 10′ showingrelative positions of the member 12, fluid handling assembly 14, andimaging assembly 15 in an enclosure 17. Like perfusion device 10, theimaging assembly 15 in perfusion device 10′ is disposed proximate themember 12. Member 12, light source 122 and the objective of microscope120 can be disposed relative to one another such that the light source122 can illuminate the channel 18 and the microscope 120 can magnify andresolve the thrombus formation in channel 18 where the thrombusformation had been previously fixed and stained within the channel 18 bythe image enhancing agents as previously described. In Köhlerillumination, the light source 122 illuminates the fixed and stainedthrombus formation. Light beams passing through the thrombus formationare refracted and captured in the object lens of the microscope 120. Thelenses of the microscope 120 resolve and magnify the image of thethrombus formation with sufficient contrast so as to enable analysis ofthe formation.

In order to capture the image of the thrombus formation in the channel18, imaging assembly 15 can also include a camera 124, shownschematically in FIG. 2D. More specifically, imaging assembly 15 caninclude a CCD camera 124 for converting the light image of the thrombusformation to a fixed digital data image. The digital data image can bestored to read/write digital medium 137 in, for example, a hard drive ofa computer or alternatively a networked data storage device. As inperfusion device 10, camera 124 of perfusion device 10′ can preferablyinclude a microscopic zoom lens to eliminate the need for the separatemicroscope 120. Alternatively, camera 124 can be interfaced withmicroscope 120 to digitally capture the image of the thrombus formation.

Alternative light contrasting techniques can be employed to image thethrombus formation as are known to one of ordinary skill in the art ofmicroscopy. Such techniques include: (i) Oblique illumination; (ii)polarization; (iii) phase contrast; and (iv) differential interferencecontrast.

The digital image data of thrombus formation captured by digital camera124 in either embodiment of perfusion device 10, can be stored,displayed and printed or otherwise processed to quantify certain aspectsof the thrombus formation, for example, the volume of thrombusformation. Perfusion device 10 can include an analyzer 16 having aprocessor 132 including an interface 134 for receiving and readingdigital image and non-image data of the thrombus formation.

Processor 132 can be a computer 136 having serial connection to digitalcamera 124 to receive the digital image data. The camera 124 may beconnected to computer 136 by a firewire connection for rapid digitalimage data transfer. Alternatively, computer 136 can have a disk driveas is known in the art for receiving and reading the digital image datastored to a portable read/write recording medium 125 of the camera 124.Processor 132 can convert the digital image data to pixel data in amanner known to one of ordinary skill in the art. Pixel data caninclude, for example, pixel color or pixel intensity. Processor 132 canfurther use the pixel data using at least one algorithm to correlateand/or quantify an aspect of the thrombus formation, i.e., the volume ofthrombus formation.

Preferably, computer 136 can include executable software or computerprogram 140 capable of running the algorithm to read the digital imagedata and convert it to pixel data to calculate and display thequantifiable aspects of thrombus formation. The computer program 140 canbe written and customized using known data acquisition software, forexample, LabView software. The pixel data determined by program 140 canbe correlated to thrombus formation in accordance with user selectedneeds. For example, pixel data indicating dark colors may be correlatedto indicate the presence of thrombus formation; therefore, largeclusters of dark colored pixel data indicate the presence of a highconcentration of thrombus formation. Alternatively, program 140 may beconfigured such that a cluster of light colored pixel data indicates thepresence of thrombus formation. The pixel data can be used to displaythe image of the thrombus formation to a display device, for example, acomputer monitor or for printout by a computer printer.

As previously described, perfusion device 10 and imaging assembly 15 caninclude a non-imaging fluorescence photodetector 127, for example, aphotodiode or photomultiplier which for converting the fluorescenceintensity of the platelets aggregated in the field of view to anelectrical signal or other non-imaging data. In perfusion device 10, acomputer 136 may be provided having software program 140 including analgorithm which can process non-imaging data received from thephotodetector 127. The software program 140 can be for example, LabViewsoftware including an analog to digital converter for reading theelectrical signal. The software program 140 can integrate the capturedfluorescence intensity over the entire field of view to give a thrombusformation curve 190 as is schematically shown in FIG. 2A. The curve 190and its data can be further processed by program 140 to provide atemporal evolution of the volume of thrombus formation in the channel 18and/or other quantifiable characteristics of thrombus formation.

Shown in FIGS. 2A and 2C is the analyzer 16 of FIG. 2 being a computer136 preferably disposed proximate the imagining assembly 15 to permitimmediate correlation of either (i) the digital image data or (ii) thenon-imaging data as it relates to the thrombus formation. The data canbe stored to the local read/write memory or hard drive of the computer136. However, alternatively, analyzer 16 can be completely separatedfrom the imaging assembly 15 and perfusion device 10. In one embodiment,analyzer 10 can include a stand alone computer 136 including a softwareor computer program 140 with at least one algorithm as previouslydescribed. Bundled detector or digital image data of blood assays can bedelivered to computer 136 for analysis. For example, bundled digitaldata image files can be stored on a read/write recording medium 125 ofimaging assembly 15 in one location and downloaded for analysis on thecomputer 136 in another location and stored to a data storage device ormedium 137 in the same or different location. The digital image datafiles can be read from the portable read/write recording medium 125using a disc drive as is known in the art. Alternatively, the digitalimage data files can be stored on a server 137, for example, on a localor wide area network, for example, on an intranet or the Internet.Permitting bundled data files concerning the thrombus formation to bestored for later analysis permits for high volume blood assays andimaging to be performed without having to run the thrombus formationanalysis in sequence with the imaging.

Referring now to FIG. 5, program 140 may include additional algorithmsto control other features of perfusion device 10, 10′ including animaging control algorithm 152 for controlling the imaging assembly 15and a fluid control algorithm 154 for controlling the delivery of fluidsto the channel 18 of member 12 or directly to the blood sample. Forexample, the imaging control algorithm 152 can be configured to controlthe exposure times and setting of camera 124 of imaging assembly 15,wherein the computer 136 and the camera 124 preferably communicate via afirewire interface. Alternatively, algorithm 152 can be configured tocontrol any of the previously described operations of the imagingassembly 15. In another example, the fluid control algorithm 154 can beconfigured to control the off/on function or the variable flow rate ofpump 106. Moreover, in assays utilizing multiple channel 18 embodimentsof member 12, the fluid control algorithm 154 can be configured to varythe flow parameters from channel to channel. In addition, algorithm 154can be configured to control, for example, the sequencing or off/ondelivery of the image enhancing agents used in the fluid handlingassembly 14. Fluid handling assembly 14 and imaging assembly 15 can becontrolled by using an appropriate interface between the computer 136executing program 140 and its algorithms 152, 154 and the equipment tobe controlled. The delivery of the image enhancing agents, in terms ofeither volumetric or sequential control, can be automated by a fluidcontrol algorithm or system 154 interfaced with liquid handling assembly14. For example, referring again to FIGS. 4E and 4H, microchip member 12can include the requisite fluid and electrical/electronic interfacesknown to one of ordinary skill in the art for connection to the bloodsample source, imaging agents source, fluid handling assembly 14, orfluid control algorithm 154. It is to be understood that liquid ports30, 32 and 34, fluid handling assembly 14 and fluid control algorithm154 can be configured so as to deliver any agent needed for the purposeof the blood assay.

Thus to practice the methods of the present invention, a perfusiondevice such as perfusion device 10 can be operated in the followingmanner. Member 12 can be prepared by providing coating 25 on at leastone of the transparent surfaces 26 defining channel 18 in order toinitiate and promote thrombus formation therein. Depending on theconfiguration of member 12, as described above, member 12 can bepre-coated with the coating 25, for example, on the upper surface 56 ofthe member 12′ having an adjusting tube member 60. Alternatively, member12 can be manually coated with the coating 25 prior to running theassay, for example, using micro-capillary tube member 12. Member 12 isthen assembled based upon its construction, as previously described, andinserted into the socket 38 of perfusion device 10 for secure holdingand orientation relative to the remaining components of the perfusiondevice 10. Any necessary tubing, for example silastic tubing, isprovided to connect the blood sample with the member 12 and the fluidhandling assembly 14. Additionally, a rinsing buffer of, for example, asaline mixture can also be run through the tubing of perfusion device 10to avoid air from developing in the piping system.

In one embodiment, the blood sample is treated with an anticoagulant. Inanother embodiment, the anticoagulant is selected from the groupconsisting of citrate, PPACK and heparin. If thrombus formation isimaged using kinetic or time lapse imaging of the formation, the bloodsample may be labeled with a fluorescent agent and slightlyanti-coagulated with a small amount of anti-coagulant, for example,heparin, Ppack, citrate, EDTA, factor Xa inhibitor or any otheranti-coagulant known in the art, while in the reservoir and prior toperfusion through member 12. In other embodiments, the blood sample isnot treated with an anticoagulant.

In one embodiment, fluid handling assembly 14 uses vacuum pressure todraw the fluorescent blood sample through the channel 18 of member 12.Specifically, fluid handling assembly 14 includes a syringe pump 106having a known flow rate so as to move the sample of blood through thechannel 18 having a cross-sectional area 24 of preferably knowndimensions at a desired shear rate. In embodiments wherein perfusiondevice 10 includes a computer 136, this may include running a softwareprogram 140 including algorithm 154 in conjunction with a userinterface. A user can use controls to set the flow rate of fluidhandling assembly 14 or pump 106 to move the blood sample at a desiredshear rate. The fluid handling assembly 14 operates to draw the bloodthrough channel 18 of member 12 for a period of time sufficient for theblood to react with the coating in channel 18 and initiate thrombusformation in the channel 18. The period of time the fluid handlingassembly 14 operates to move the blood sample through the channel 18 canbe controlled by algorithm 152. In one embodiment, whole unfractionatedblood is pumped through the perfusion device. In another embodiment, therate of blood deposit is sufficient to determine the extent of aspirinresponsiveness within approximately 5 minutes of the start of perfusion.

Referring back to FIGS. 2A and 2B, during perfusion of the blood samplethrough the member 12 and as previously described, the imaging assembly15 repeatedly images the channel 18 at defined intervals to capture theevolving thrombus formation. Member 12 may be maintained in socket 38 ofperfusion device 10 for microscopy imaging by the imaging assembly 15 inaccordance with the microscopy techniques described above. Preferably,computer 136 having software program 140 including algorithm 152 andcontrols of user interface 142, operate the LED and preferably camera124 including microscopic zoom lens to capture digital images of thethrombus formation under light microscopy. Alternatively, lightmicroscope 120 is operated by computer 136 to bring the magnificationand resolution of the thrombus formation into focus and coupled camera124 captures the digital data image. The computer 136 and program 140can additionally be configured to translate socket 38 in order to bringthe thrombus formation into focus for imaging. Camera 124 can beemployed with a frame rate of about 2 frames per second to capture atime-lapse image of thrombus formation. The imaging assembly 15 can takean image of thrombus formation at various points along the longitudinalaxis A-A of channel 18. The time-lapse digital image data is then storedto a read/write recording medium, for example, the data storage device137. Member 12 can then be removed from socket 38 and can be replaced bya new member 12 for running a new assay.

Once again, the user using the computer 136 having software program 140,algorithm and user interface 142 can select the digital image data filesfor analysis. The program 140 uses the algorithm 138 to process thedigital image data so as to generate the pixel data. For each digitaldata image, mean pixel values, mean pixel intensities are determined andthe values are displayed as outputs 146, 148. A graphic of the thrombusformation is provided in display 144 of user interface 142. The pixeldata is correlated to the volume of thrombus formation and reported tothe user for use in identifying and treating aspirin non-responsivepatients or adjusting the anti-thrombogenic therapy.

The processor 132 or computer 136 can be configured to utilize availableconventional software applications capable of reading a digital dataimage and converting it to visual scale data. The visual scale data canbe further correlated to the quantifiable aspects of thrombus formation.For example, computer 136 can be configured to run a softwareapplication 140 capable of reading static digital image data andconverting it to mean grayscale data, where the mean grayscale data is ameasure of intensity or darkness of the blood sample imaged in thechannel 18. Any scale can by used to measure the intensity or darkness,for example, a mean grayscale can range from zero to about 255, whereinzero is black and 255 is white. Digital image data read to have a lowmean grayscale score can indicate the presence of thrombus formation.Alternatively, the grayscale may be applied inversely such that a highgrayscale score indicates thrombus formation. Software application 140can be commercially available software, for example, PHOTOSHOP™,configured to run on a processor 132 or computer 136. Alternatively,grayscale level measurements may be performed manually.

In addition or alternatively to the camera 124, a non-imagingphotodetector 127 can be provided to pick up the fluorescence intensityfrom aggregated platelets in the channel 18 to generate an electricalsignal. The signal from the photodetector 127 can be read by thecomputer 136 having software 140 with imaging algorithm for correlatingthe fluorescence non-imaging data to the temporal evolution of thevolume of thrombus formation or any other temporal and quantifiablecharacteristic of the thrombus formation. Moreover, the user can useinterface 142 to graphically display the fluorescence data correlated tothe quantifiable attributes of the thrombus formation, for example suchas the graph shown in FIG. 2A.

Photodetector 127 can be configured with computer 136 so as to capturetime-lapse or temporal evolution images of light emitted from a thrombusformation, coagulation or any cellular movement in member 12 and displaythe image as a digital image data on a frame by frame basis. The imagingalgorithm may be configured to read a single frame of displayed digitalimage data from photodetector 127 as an array of pixels, for example1024×768 pixels, each pixel having a quantifiable pixel intensity.Because of the relative position of the photodetector 127 to themicroscope objective of microscope 120 in imaging assembly 15, lightemitted from the thrombus formation in member 12 and received by thephotodetector 127 becomes diffused and appears as background. As aresult, the imaging algorithm includes a first aspect, background orcontrol subtraction step for removing a background or control image soas to isolate the thrombus image for quantifiable measurement.

Thus in one embodiment, the assay enables the investigator to evaluatethrombotic process under arterial shear rates in less than 15 minutesafter blood draw. The simplicity of the perfusion chamber allows formultiple parallel experiments as the thrombogenic surface is depositedinside a regular glass capillary tube. The system is easy to use as only2 tubings are needed: a proximal tubing to connect the blood sample tothe capillary, and a distal tubing which connects the capillary to thepump. A limited amount of blood is required to reconstitute arterialshear rate conditions (typically 4 ml of blood for 1 perfusion chamber).Whole blood (non-anticoagulated, PPACK-, or citrate-anticoagulated humanblood) can be perfused through coated capillaries at arterial shearrates for 4 minutes.

Comparing and Subtracting Control Levels

Within the method of the present invention, the status of aspirinresponsiveness is determined by measuring the level of blood depositedin the channel and comparing the measured level of deposited blood witha control or threshold level of deposited blood. In certain embodiments,the control or threshold level is selected from the group consisting of(1) a level measured using a sample of blood from said subject which hasbeen treated with aspirin; (2) a level measured using a sample of bloodfrom said subject which has been contacted with a thromboxane A2receptor antagonist; and (3) a level previously determined to correspondto a status of aspirin responsiveness. This comparison can be donemanually as described above or the control levels may be electronicallysubtracted.

In subtraction step, the 1024×768 array of pixels may be divided into asubsection array of pixels, for example, a subsection array of 32×32pixels. For each subsection of the array, a threshold value of pixelintensity is determined. This threshold value may be defined by thebackground intensity of the subsection array or by a previously measuredvalue. In order to reduce or eliminate the noise content of the digitalimage, each subsection is subjected to a low-pass filtering process. Thelow-pass filter preferably includes a cut-off frequency of 30% themaximal spatial frequency contained in the image data. A threshold isdetermined for the low-pass filtered image of each subsection. Morespecifically, any pixels having an intensity of less than a given valuecorresponding to adherence of a platelet, for example 10, are preferablyset to zero.

The imaging algorithm includes a second aspect or area calculation.Following determination of the threshold for each subsection, areacalculation includes taking the balance of pixels with an intensitygreater than zero and resetting their intensity value preferably to one.The sum of the pixels in the subsection array define the thrombus areain units of (pixel dimension).

The imaging algorithm includes a third aspect or volume calculation 186.Following determination of the threshold for each subsection, volumecalculation 186 includes taking the balance of pixels with an intensitygreater than zero and taking the summation of those intensity values todefine a thrombus volume measured in (pixel dimension)2×pixel intensity.Dividing the thrombus volume by the thrombus area can provide a meanthrombus height value.

The imaging algorithm includes a fourth aspect or perimeter calculation.Following determination of the area calculation, perimeter calculationincludes taking the image of pixels, each having an intensity of one,and passing it through a high-pass filtering process. The high-passfilter includes a cut-off frequency of preferably about 50% of themaximum spatial frequency contained in the threshold image. Combiningthe perimeter calculation with the area calculation can provideinformation about the shape of the thrombus formation.

In an alternative of embodiment imaging algorithm, imaging algorithm caninclude a first aspect or segmentation process, and second aspect ornoise reduction process 184′, and a third aspect or watershed separationprocess. Wherein photodetector 127 preferably produces a grayscaledigital image data composed of pixels of varying pixel intensity,segmentation process which includes binarizing the grayscale digitalimage by producing a histogram for a single frame of data showing pixelintensity versus number of pixels. Taking the first derivative, secondderivative or percentile method of the histogram of each image locatesdiscrete peaks in the plot. More specifically, taking the secondderivative of the initial histogram plot can reveal at least two minimapoints, although more are possible, wherein the first or lower minimumdefining a threshold pixel intensity value. The threshold value furtherdefines a cut-off for which pixels having an intensity less than thethreshold value form the background of the digital image and theremaining foreground define the thrombus formation.

Alternative methods of computing the threshold can be utilized in whicha threshold value is applied to all the images generated by theexperiment. For example, the threshold value can be determined for allthe images using Otsu's method (bimodal with equal variance), Kapur,Sahoo & Wong's method (1D entropy), or Abutaleb's method (2D entropy).For each of these methods, the threshold value was computed for theentire run of the experiment and then Gaussian smoothing was appliedbefore the threshold was applied to the corresponding images.

With the threshold determined, the noise reduction process includes afirst morphological operation in which small objects, for example, 5pixels in width, that appear in the image close together, for example,within a distance of 2 pixels between each other, the objects are mergedtogether. Next, the resultant image is subjected to a secondmorphological operator in which isolated voids appearing as white pixelsare removed. In addition or alternatively to, small objects appearingwithin larger objects of the digital image data are subject to a logicaloperation in which pixels of the original digital image data and thedigital image data produced by the first and second morphologicaloperations are ANDed to produce a single image. The resultant image issmoothed by a median filter so as to define a final threshold mask.

The original digital image is modified by subtracting the thresholdintensity value from all the pixels and applying the threshold mask tothe image, thereby discarding background pixels.

The watershed separation process is applied to the resultant image, soas to identify the individual thrombi. Pixel intensity value maxima areidentified and assigned a discrete color. Where discrete colors aresubstantially close so as to appear to merge a digital divider islocated therebetween to partition the digital images of individualthrombi. The watershed is analogized to a flooding simulation. Thedigital image is turned upside-down, so that intensity maxima correspondto watershed minima. Modeling the image as a plastic surface, thewatershed minima are imagined to define small pools in the surface withsmall holes in them. Imagining that the surface is submerged in waterwith water entering the holes such that the water level rises in thepool. Each individual pool is isolated by a dam, and anytime the poolthreatens to overflow and merge with another, a dam is built to containthe overflow.

Having identified the individual thrombi, thrombus area, volume, andperimeter can be determined. For a given image, the thrombus area isobtained by counting the number of pixels forming the individualthrombi, the volume is obtained by summing the pixel intensity valuesfor an individual thrombi, the perimeter can be obtained by counting thenumber of pixels that are on the edges of the thrombi. A time-lapseframe by frame plot of thrombi growth/decay can be provided by fittingthe volume data to a 10th degree polynomial to display the thrombiquantities.

In an alternate method in which the thrombus formation is to be imagedusing fixed end point measurement imaging, a sample of blood, preferablynon-anticoagulated blood, is provided for moving through member 12. Theblood sample can be drawn from a reservoir and perfused through member12 in a manner as previously described. Alternatively, the sample ofblood can be drawn directly from a person. A patient can be undergoinganti-thrombotic drug treatment and can be hooked up to the perfusiondevice 10 to monitor thrombosis in the patient's blood. For example, thepatient can be given a dose of medication and then immediately followingthe dosage, blood can be perfused through system 10 to determine whetherthe amount of medication is appropriate. Preferably and schematicallyshown in FIG. 2D, fluid handling portion 14 a can include the requisitetubing and fittings to draw blood from a reservoir collection vessel(not shown) in a manner well known in the art.

Once the perfusion of the blood sample through the channel 18 iscomplete, the thrombus formation can be fixed and stained for microscopyimaging. Preferably, fluid handling portion 14 b in FIG. 2D drawsimaging enhancing agents from a source (not shown). For example, thethrombus formation may be rinsed and then fixed using a solution ofeither PBS, glutaraldehyde 2.5% or PBS, PFA 4%. The fluid handlingportion 14 b can apply a toluidin blue solution to stain the thrombusformation and repeatedly rinse the channel 18 with the a rinsing buffer.The member 12 is then prepared for imaging of the thrombus formation.

Member 12 may be maintained in socket 38 of perfusion device 10 formicroscopy imaging by the imaging assembly 15 in accordance with thelight microscopy techniques using Köhler Illumination. As previouslydescribed, computer 136 having software program 140 including algorithm152 and controls of user interface 142 can translate the socket 38 andoperate the LED 122 and camera 124 including microscopic zoom lens oralternatively interfaced microscope 120 to focus and capture fixed endpoint digital images of the thrombus formation.

1. The user using the computer 136 having software program 140,algorithms and user interface 142 can select the digital image datafiles for analysis. The program 140 uses the digital image data in analgorithm to generate the pixel data. For each digital data image, meanpixel values, mean pixel intensities are determined and the values aredisplayed as outputs 146, 148. A graphic of the thrombus formation isprovided in display 144 of user interface 142. The pixel data iscorrelated to the volume of thrombus formation and reported to the userfor use in adjusting the anti-thrombogenic therapy.

In another embodiment, comparisons with control levels are done inseparate experiments. Whole blood is perfused through the channels.Platelet interactions and thrombus formation are recorded in real timewith the software. The mean fluorescence intensity parallels thekinetics of the thrombotic process, if platelets are labeled by afluorescent label such as rhodamine 6G. The kinetics of the thromboticprocess is recorded at 2 frames per second for 5 minutes and plottedversus time. This technique allows a physician or investigator todetermine whether a drug exhibits an anti-adhesive or anti-aggregatory(platelet-to-platelet interactions) activity and its effects on thrombusstability.

Thus once the profiles of the platelet/leukocyte aggregates areestablished, the effects of aspirin can be evaluated. For example, anaspirin dose response study can be comparably performed (by addition ofliquid aspirin, Aspegic). Similarly, other antithrombotic agentsantithrombotic drugs (GP IIb-IIIa inhibitors (integrilin), directthrombin inhibitor (Angiomax), P2Y12 antagonism(5-chloro-N-[({4-[7-fluoro-6-(methylamino)-1-oxoisoquinolin-2(1H)-yl]phenyl}amino)carbonyl]thiophene-2-sulfonamide))can be evaluated in order to obtain a mean thrombotic profile for eachtreatment. This method can also be tested with blood treated withcombinations of treatments such as P2Y₁₂ inhibitor+epinephrine, in thepresence or absence of aspirin. A schematic representation of thechamber (Aspirin-Chip) is shown in FIG. 10.

Thus in one embodiment, the perfusion device provides real timemeasurements of (1) the thrombotic profile of aspirin effects on top ofP2Y₁₂ antagonism in a collagen-coated perfusion chamber assay underarterial shear rates and (2) arachidonic acid/shear-induced plateletaggregation assay in whole blood under high shear rates.

In another embodiment, the invention described herein provides a methodfor utilizing a single apparatus that will evaluate: 1) the thromboticprofile of aspirin effects on top of P2Y₁₂ antagonism in acollagen-coated perfusion chamber assay under arterial shear rates; 2)arachidonic acid/shear-induced platelet aggregation assay in whole bloodalso under high shear rates. One main advantage of this technique isthat it will enable the investigator to assess the variability of theaspirin resistant phenotype and its dose dependence. A lack of efficacyof aspirin in both assays will be correlated with aspirin resistance. Amajor limitation of the PFA-100 device as well as classical perfusionchambers is the lack of real time qualitative assessment of bothplatelet adhesion and thrombus formation. Another advantage of thistechnique is that it is insensitive to the presence of therapeuticamounts of platelet ADP-receptor antagonists in the subject'sbloodstream. Thus in another embodiment, the method is insensitive tothe presence of therapeutic amounts of platelet ADP-receptor antagonistsin the subject's bloodstream.

Methods of Treatment/Administration

One aspect of the present invention is identifying subjects with aspirinresistance so that they qualify for treatment with a thromboxane A2receptor antagonist. Thus in one embodiment, the present inventionprovides a method of qualifying a subject for treatment with athromboxane A2 receptor antagonist, comprising:

(a) perfusing a blood sample from said subject through a channel in aperfusion device, wherein the channel has a coating which produces ablood deposit when exposed to blood and the perfusion device comprises apump coupled to the outlet end of the housing to draw the blood throughthe channel at a desired shear rate, producing a blood deposit, andwherein said blood sample is treated with an amount of a platelet ADPreceptor antagonist sufficient to inhibit thrombosis at leastapproximately 20% relative to an untreated sample; and

(b) wherein said blood sample is treated with an amount of aspirinsufficient to cause at least an approximately 50% inhibition ofthrombosis in a blood sample relative to an untreated sample; and

(c) wherein a subject is qualified for treatment with a thromboxane A2receptor antagonist if less than approximately 50% inhibition ofthrombosis is observed in said blood sample relative to an untreatedsample.

Once a subject has been identified as an aspirin non-responder using theabove methods, they may be treated with a thromboxane A2 receptorantagonist, either alone or in combination a platelet ADP-receptorantagonist. Examples of thromboxane A2 receptor antagonists include butare not limited to Terbogrel, Ridogrel, Ramatroban, Seratrodast,Ozagrel, Ifetroban, BM-531 and S18886. Examples of platelet ADP-receptorantagonists include, but are not limited to Plavix and clopidogrel.Preferably, the platelet ADP-receptor antagonist is Plavix. Methods forpreventing or treating thrombosis in a mammal by administering to themammal a therapeutically effective amount of these compounds, alone oras part of a pharmaceutical composition of the invention as describedabove can be done following procedures known to those of skill in theart.

The methods of the invention may be utilized in vivo, ordinarily inmammals such as primates, (e.g., humans), sheep, horses, cattle, pigs,dogs, cats, rats and mice, or in vitro.

Subjects (typically mammalian) in need of treatment by the methods ofthe invention may be administered dosages that will provide optimalefficacy. The dose and method of administration will vary from subjectto subject and be dependent upon such factors as the type of mammalbeing treated, its sex, weight, diet, concurrent medication, overallclinical condition, the particular compound employed, the specific usefor which the compound or pharmaceutical composition is employed, andother factors which those skilled in the medical arts will recognize.

Therapeutically effective dosages may be determined by either in vitroor in vivo methods. For each particular compound or pharmaceuticalcomposition of the invention, individual determinations may be made todetermine the optimal dosage required. The range of therapeuticallyeffective dosages will be influenced by the route of administration, thetherapeutic objectives and the condition of the patient. For injectionby hypodermic needle, it may be assumed the dosage is delivered into thebodily fluids. For other routes of administration, the absorptionefficiency must be individually determined for each compound by methodswell known in pharmacology. Accordingly, it may be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect.

The determination of effective dosage levels, that is, the dosage levelsnecessary to achieve the desired result, i.e., platelet ADP receptorinhibition, will be readily determined by one skilled in the art.Typically, applications of a compound or pharmaceutical composition ofthe invention are commenced at lower dosage levels, with dosage levelsbeing increased until the desired effect is achieved. The compounds andcompositions of the invention may be administered orally in an effectiveamount within the dosage range of about 0.01 to 1000 mg/kg in a regimenof single or several divided daily doses. If a pharmaceuticallyacceptable carrier is used in a pharmaceutical composition of theinvention, typically, about 5 to 500 mg of a compound is combined with apharmaceutically acceptable carrier as called for by acceptedpharmaceutical practice including, but not limited to, a physiologicallyacceptable vehicle, carrier, excipient, binder, preservative,stabilizer, dye, flavor, etc. The amount of active ingredient in thesecompositions is such that a suitable dosage in the range indicated isobtained.

In another embodiment the aspirin non-responder is a subject having anaspirin responsiveness of less than or equal to 50% of the value foraspirin responsiveness. Thus in one embodiment, the present inventionprovides a method of treating an aspirin non-responsive subjectcomprising:

(a) identifying a subject as an aspirin non-responder using any of theabove methods, wherein an aspirin non-responder is a subject having anaspirin responsiveness of less than or equal to 50% of the value foraspirin responsiveness; and

(b) administering to the aspirin non-responsive subject a thromboxane A2receptor antagonist. In another embodiment, the thromboxane A2 receptorantagonist is selected from the group consisting of Terbogrel, Ridogrel,Ramatroban, Seratrodast, Ozagrel, Ifetroban, BM-531 and S18886. Inanother embodiment, the present invention provides a method of treatingan aspirin non-responsive subject comprising the steps of:

identifying a subject as an aspirin non-responder according to any ofthe above methods; and

administering to the subject a thromboxane A2 receptor antagonist incombination with a platelet ADP-receptor antagonist. In one embodiment,the thromboxane A2 receptor antagonist is selected from the groupconsisting of Terbogrel, Ridogrel, Ramatroban, Seratrodast, Ozagrel,Ifetroban, BM-531 and S18886. In another embodiment, the plateletADP-receptor antagonist is selected from the group consisting of Plavixand clopidogrel. In another embodiment, the platelet ADP-receptorantagonist is Plavix.

The following examples are offered by way of illustration only and arenot intended to limit the scope of the claimed invention. Numerousembodiments of the invention within the scope of the claims that followthe examples will be apparent to those of ordinary skill in the art fromreading the foregoing text and following examples.

EXAMPLES Example 1

Evaluation of RPFA-ASA and PFA-100

The effects of aspirin (275 μM added to the blood in vitro) and 50%inhibition of P2Y₁₂ (corresponding to the extent of inhibition achievedin patients under Plavix treatment) were evaluated in the RPFA-ASA(n=5). A direct P2Y₁₂ antagonist(5-chloro-N-[({4-[7-fluoro-6-(methylamino)-1-oxoisoquinolin-2(1H)-yl]phenyl}amino)carbonyl]thiophene-2-sulfonamide)is used in vitro at a concentration that inhibits 50% of ADP-inducedplatelet aggregation in PRP as Plavix cannot be used in vitro (it needsto be metabolized by the liver to generate the active metabolite). Theresults show that aspirin effects were smaller than that obtained with50% inhibition of P2Y₁₂ (FIG. 1, blood donor A and B) for all 5 blooddonors. Also, one donor presented an aspirin-like phenotype withoutaddition of aspirin or aspirin uptake (donor B). Since the actualanti-platelet regimen consists of a combination of aspirin and P2Y₁₂antagonist, the RPFA-ASA assay cannot detect aspirin response on top ofP2Y₁₂ inhibition.

The effects of aspirin (normal volunteers were drug free for 2 weeksprior to 325 mg/d ASA for 3 days) and 50% inhibition of P2Y₁₂ onarachidonic acid-induced platelet aggregation in PRP, TxB2 generation,PFA-100 with both Collagen/Epinephrine (CEpi) and Collagen/ADP (CADP)cartridges were evaluated. Arachidonic acid-induced platelet aggregationand TxB2 generation were abolished by aspirin treatment compared withbaseline for all 5 donors. In the PFA-100 model, the effect of aspirinis revealed by an increase in closure time in the CEpi cartridge, withan unchanged value for the CADP cartridge. All donors gave the expectedprolongation of the CEpi cartridge after aspirin treatment. In addition,2 out of 5 donors presented unusual phenotypes (FIG. 6). In donor 1,aspirin increased the CADP closure time, whereas in donor 2, P2Y₁₂antagonism increased closure time of the CEpi cartridge above the limitdefined by the manufacturer for aspirin effect.

These preliminary studies highlight differences in the plateletmonitoring devices in a normal population with both RPFA-ASA andPFA-100, and may likely be more variable in clinical studies withpatients, potentially leading to multiple misinterpretations. Inaddition, none of the assays gave a qualitative real time measurement ofthe thrombotic process to reveal full or partial inhibitory effects ofaspirin in vivo. Finally, whether the PFA-100 correlates with futureclinical events is uncertain (Gum, P. A. et al., J Am Coll Cardiol,41(6):961-5 (2003)).

Example 2

Perfusion Chamber Assays

In one example of the perfusion chamber assay of the invention, theassay enables the investigator to evaluate thrombotic process underarterial shear rates in less than 15 minutes after blood draw. Thesimplicity of the perfusion chamber allows for multiple parallelexperiments as the thrombogenic surface is deposited inside a regularglass capillary tube. The system is easy to use as only 2 tubings areneeded: a proximal tubing to connect the blood sample to the capillary,and a distal tubing which connects the capillary to the pump. A limitedamount of blood is required to reconstitute arterial shear rateconditions (4 ml of blood for 1 perfusion chamber). Whole blood(non-anticoagulated, PPACK-, or citrate-anticoagulated human blood) isperfused through fibrillar type III collagen-coated capillaries atarterial shear rates for 4 minutes.

At the end of the perfusion, the capillary is rinsed for 20 sec andfixed with 2.5% phosphate buffered glutaraldehyde for 1 minute.Capillaries are stained for 45 sec with toluidine blue, then rinsed withbuffer “C”. An En Face picture located 5 mm downstream of the proximalpart of the capillary is taken, and the grey level of each thrombus orplatelets located in a window 400 μm long×400 μm wide is measured(Simple PCI software, Compix Inc. Imaging System). Thrombotic depositswere then embedded in Epon, subsequently cross sectioned (at the samelocation used for measurement of the grey level) and their mean thrombusvolume determined. Mean grey level is then plotted against thecorresponding mean thrombus volume. A thrombotic profile curve isobtained by titrating with the GP IIb-IIIa antagonist eptifibatide (FIG.7). The same profile can be generated for the rectangular perfusionchamber and utilized for a rapid measurement of the effects of differentdrugs on the arterial thrombotic process.

Using this technique, the antithrombotic effects of aspirin and P2Y₁₂inhibitors are observed to synergize on collagen. The same technique maybe used to show that P2Y₁₂-deficient mice are protected from vascularocclusion following FeCl₃ injury of mesenteric arteries. This phenotypeis rescued by the injection of epinephrine in vivo (Andre, P. et al., JClin Invest, 112(3):398-406 (2003)). Similarly, epinephrine was able tocorrect the phenotype of P2Y₁₂-deficient mice in a collagen-coatedperfusion chamber (epinephrine stimulates the Gz pathway thatcompensates for the blockade of the P2Y₁₂-coupled Gi pathway).

Example 3

Collagen-Coated Perfusion Chamber Assay to Measure Aspirin Resistance inWhole Blood.

Platelets are labeled in whole blood with Rhodamine 6G and used to showthat aspirin and thrombin inhibitors synergize with P2Y₁₂ antagonism(Andre, P. et al., Circulation, 108(21):2697-703 (2003)). After bloodcollection into either citrate or PPACK (4 ml are needed per perfusionchamber) Rhodamine 6G (0.2 mg/mL) is added to the blood and incubated at37° C. for 5 minutes. Collagen-coated rectangular glass capillaries(Vitrocom) are mounted on the stage of a fluorescent microscope andilluminated under light, UV, or both. Whole blood is perfused at1000/sec through the capillaries. Platelet collagen interactions andthrombus formation are recorded in real time with the Simple PCIsoftware. The mean fluorescence intensity parallels the kinetics of thethrombotic process, because all platelets are labeled by rhodamine 6G.The kinetics of the thrombotic process is recorded at 2 frames persecond for 5 minutes and plotted versus time. This technique allows aphysician or investigator to determine whether a drug exhibits ananti-adhesive or anti-aggregatory (platelet-to-platelet interactions)activity and its effects on thrombus stability. Experiments areperformed with different antithrombotic drugs (GP IIb-IIIa inhibitors(integrilin), direct thrombin inhibitor (Angiomax), P2Y₁₂ antagonism(5-chloro-N-[({4-[7-fluoro-6-(methylamino)-1-oxoisoquinolin-2(1H)-yl]phenyl}amino)carbonyl]thiophene-2-sulfonamide))and notably aspirin in order to obtain a mean thrombotic profile foreach treatment. This method is then tested with blood treated with P2Y₁₂inhibitor+epinephrine, in the presence or absence of aspirin.

Most ACS patients eligible for percutaneous coronary intervention (PCI)are under Plavix therapy. Since the level of P2Y₁₂ inhibition varies inpatients (depending on the loading doses of Plavix and the time ofadministration) a full blockade of the P2Y₁₂ receptor is achieved with5-chloro-N-[({4-[7-fluoro-6-(methylamino)-1-oxoisoquinolin-2(1H)-yl]phenyl}amino)carbonyl]thiophene-2-sulfonamide,a direct P2Y₁₂ inhibitor, in vitro. P2Y₁₂ antagonism is overcome bystimulation of the G protein z (Gz) pathway with epinephrine added tothe whole blood prior to perfusion. This reveals the aspirinantithrombotic activity in all patients. Trisodium citrate is used inthis example as anticoagulant because it reveals aspirin effects inperfusion chamber assays (Baumgartner, H. R. et al., Thromb Haemost,35(1):124-38 (1976)).

Fibrillar type III collagen has been chosen in this example for thethrombogenic material as the effects of aspirin can be observed inperfusion chambers coated with fibrillar collagen under arterial shearrate conditions. In addition, increased responsiveness to collagen hasbeen proposed to contribute to aspirin resistance (Zimmermann, N. etal., J Thorac Cardiovasc Surg, 121(5):982-4 (2001); Kawasaki, T., etal., Stroke, 31(3):591-5 (2000)). Preparation of type III fibrillarcollagen requires dialysis against phosphate buffer for 40 hours.Therefore, collagen Horm (a mixture of different fibrillar collagens)can be used as alternative in this system since it is commerciallyavailable and does not require extensive preparation for coating.

Volunteers from a blood donor program can be used to firmly establishthe concentration of epinephrine needed for correction of the P2Y₁₂antagonism and to evaluate aspirin effects on P2Y₁₂antagonism+epinephrine background.

FIG. 8 shows the real time kinetics of thrombus formation in a perfusionchamber perfused with either untreated PPACK-anticoagulated blood (blackcircles and black line) or 2.5 μM Integrilin (GP IIb-IIIaantagonist)-treated PPACK-anticoagulated blood.

A perfusion chamber assay on type III collagen (DMSO (vehicle) treated(a), full P2Y₁₂ antagonism (b), full P2Y₁₂ antagonism with epinephrine20 μM(c)) have been performed before and 3 days after a daily dose ofaspirin (325 mg). Results of the 2 blood donors are presented in FIG. 9.The data in FIG. 9 demonstrate that aspirin efficacy can be evaluated inabsence or presence of P2Y₁₂ antagonism.

Example 4

An Arachidonic Acid-/Shear-Induced Platelet Aggregation Assay in WholeBlood

Platelets are labeled in whole blood with Rhodamine 6G as describedabove. Following addition of AA, blood will be perfused through 2successive chambers which generate high (1500/sec) and low (50/sec)shear rates. The high shear rate induces platelet activation andaggregation is created by a sudden decrease in capillary diameter. Thelow shear rate area allows for the real time evaluation/quantificationof the size of the platelet/platelet, platelet/leukocyte aggregates thatwill pass and/or adhere via the Simple PCI software or similar softwarecapable of evaluating the size of the flowing thrombi.

In this example, a concentration of AA is used which will lead tothrombus formation in whole blood without occluding the proximal part ofthe channel. In other examples, the shear rates for both the proximaland distal parts of the channel are idealized.

Large mixed platelet/leukocyte aggregates are detectable in the lowshear area (observable after less than 30 seconds) when whole blood istreated with 0.8 mM arachidonic acid and perfusion through a smalluncoated circular capillary at 1700/sec followed by a larger capillaryat 80/sec. These aggregates are not present in the absence of AA, andare prevented by the use of aspirin and a mixed TP/thromboxane synthaseinhibitor (Terbogrel).

Once the profiles of the platelet/leukocyte aggregates are established,the effects of aspirin are evaluated. For example, an aspirin doseresponse study can be performed (by addition of liquid aspirin,Aspegic). Similarly, other antithrombotic agents (direct thrombininhibitors (Angiomax), P2Y₁₂ antagonists(5-chloro-N-[({4-[7-fluoro-6-(methylamino)-1-oxoisoquinolin-2(1H)-yl]phenyl}amino)carbonyl]thiophene-2-sulfonamide)can be evaluated. A schematic representation of the chamber(Aspirin-Chip) is shown in FIG. 10.

Example 5

TP and PGD2 Antagonism Induces Dethrombosis

Evaluation of the ability of thromboxane receptor (TP) antagonists todestabilize preformed thrombi was performed as followed in the real timethrombosis perfusion chamber assay (monitoring of fluorescently labeledplatelets onto collagen coated surface). Untreated samples of blood werefirst perfused through the collagen-coated perfusion chamber for 210seconds at 1500/sec. A second perfusion of blood treated with either(FIG. 11A); saline or 2.4 μM eptifibatide (therapeutic dose), 2-MeSAMP(a direct P2Y₁₂ antagonist at 100 μM a dose that totally blocksaggregation induced by ADP); or direct TP antagonists (FIG. 11B);SQ29548 (1 μM) and Ifetroban; 0.3 μM) immediately followed the firstperfusion. Thrombus growth was stopped by the addition of eptifibatidewith no apparent destabilization (FIG. 11A; mean slope after firstperfusion (mSAFP): saline, 2.7±0.3 n=7; eptifibatide, 0.17±0.22, n=7,p<0.05 vs saline). Addition of 100 μM 2-MeSAMP caused suddendestabilization of preformed thrombi resulting in marked dethrombosis(FIG. 11A; mSAFP=−1.8±0.2, n=7, p<0.001 vs eptifibatide and p<0.0001 vssaline). TP antagonism lead to a strong destabilization of preformedthrombi (FIG. 1B; Ifetroban; mSAFP=−2.5±0.6; n=6, p<0.0001 vs saline).Aspirin alone (saline control) induced a slow and moderatedestabilization of the thrombi (FIG. 12A; mSAFP=−1.07±0.3). Addition of2MeSAMP and eptifibatide to aspirinated blood immediately arrestedthrombus growth and induced sudden dethrombosis (p<0.05 vs respectivemonotherapies and aspirin alone). FIG. 12A; 2-MeSAMP 100 μM,mSAFP=−4.1±0.7; n=7; eptifibatide (mSAFP=−2.6±0.9; n=7). The presence ofaspirin abolished the destabilization effects of Ifetroban (FIG. 12B).Since Cox-1 inhibition is known to prevent the synthesis of secondmessengers that are pro-aggregatory (TxA2) but also anti-aggregatory(i.e. PGD2), whereas TP antagonism only affects the binding of TxA2 toTP, endogenous inhibitory messenger like PGD2 in untreated samples ofblood but not in aspirin-treated blood may account for the strongerdestabilization activity of inhibitors of the TP receptor. In order toevaluate a possible role for PGD2 in mediating thrombus destabilization,aspirinated blood was perfused in presence of PDG2 over preformedaspirinated thrombi. This resulted in a significant, spontaneousreversal of thrombosis (FIG. 13; profiles of platelet deposition (meanpixel value) in collagen-coated perfusion chamber. Representative of 3independent experiments).

This indicates that signaling pathways leading to increased cAMP levels(via inhibition of P2Y₁₂ or agonist activity of PDG2) confers thrombusinstability, and that TP antagonists could be used for treatment of AMIin combination with P2Y₁₂ antagonists but not in combination withaspirin. Thus in one embodiment, thromboxane A2 receptor antagonistinduces dethrombosis. In another embodiment, thrombosis can be inhibitedby increasing cAMP levels via P2Y₁₂ antagonism or via PGD2 stimulation.

All publications, patents, accession number, patent applications andreferences cited in this specification are herein incorporated byreference in their entirety for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations and changes to thedescribed embodiments are possible without departing from the sphere andscope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

1. A method of evaluating aspirin responsiveness in a subject,comprising: (a) perfusing a blood sample from a subject through achannel in a perfusion device, wherein the channel has a coating whichproduces a blood deposit when exposed to blood and wherein the perfusiondevice comprises a pump which draws the blood through the channel at aselected shear rate, producing a blood deposit; and (b) determining astatus of aspirin responsiveness by measuring the level of blooddeposited in the chamber and comparing the measured level of depositedblood with a control level of deposited blood, wherein said controllevel is selected from the group consisting of (1) a level measuredusing a sample of blood from said subject which has been treated withaspirin; (2) a level measured using a sample of blood from said subjectwhich has been contacted with a thromboxane A2 receptor antagonist; and(3) a level previously determined to correspond to a status of aspirinresponsiveness.
 2. The method of claim 1, wherein whole unfractionatedblood is pumped through the perfusion device.
 3. The method of claim 1,wherein the blood sample is not treated with an anticoagulent.
 4. Themethod of claim 1, wherein the blood sample is treated with ananticoagulent.
 5. The method of claim 4, wherein the anticoagulant isselected from the group consisting of citrate, PPACK and heparin.
 6. Themethod of claim 1, wherein said selected shear rate is an arterial shearrate.
 7. The method of claim 1, wherein said perfusion device furthercomprises a substantially flat transparent housing, wherein said channelextends through the housing with an inlet end for receiving a continuousflow of blood and an outlet end for discharging the blood.
 8. The methodof claim 1, wherein said coating comprises a coating selected from agroup consisting of fibrillar collagen type III, fibrillar collagen typeI, fibrinogen and tissue factor.
 9. The method of claim 1, wherein therate of blood deposit is sufficient to determine the extent of aspirinresponsiveness within approximately 5 minutes of the start of perfusion.10. The method of claim 1, wherein the measurement of the rate of blooddeposit comprises obtaining images of the deposited blood, andcalculating the levels of deposited blood from the images, wherein saidimages are obtained at least once every ten seconds during perfusion.11. The method of claim 1, wherein said coating is human type IIIcollagen and wherein said subject has been administered aspirin, furthercomprising the addition of an ADP-receptor antagonist to the sample,wherein said concentration of said antagonist in the sample issufficient to cause a 50% inhibition of platelet aggregation relative toa sample which has been treated with aspirin but not treated with saidADP-receptor antagonist.
 12. The method of claim 11, wherein saidconcentration of platelet ADP-receptor antagonist is approximately 10μM.
 13. The method of claim 1, wherein said method is insensitive to thepresence of therapeutic amounts of platelet ADP-receptor antagonists inthe subject's bloodstream.
 14. A method of treating an aspirinnon-responsive subject comprising: (a) identifying a subject as anaspirin non-responder using the method of any of claim 1, wherein anaspirin non-responder is a subject having an aspirin responsiveness ofless than or equal to 50% of the value for aspirin responsiveness; and(b) administering to the aspirin non-responsive subject a thromboxane A2receptor antagonist.
 15. A method of treating an aspirin non-responsivesubject comprising the steps of: identifying a subject as an aspirinnon-responder according to the method of claim 1; and administering tothe subject a thromboxane A2 receptor antagonist in combination with aplatelet ADP-receptor antagonist.
 16. The method of claim 14, whereinthe thromboxane A2 receptor antagonist induces dethrombosis.
 17. Themethod of claim 14, wherein the thromboxane A2 receptor antagonist isselected from the group consisting of Terbogrel, Ridogrel, Ramatroban,Seratrodast, Ozagrel, Ifetroban, BM-531 and S18886.
 18. The method ofclaim 14, wherein the thromboxane A2 receptor antagonist is selectedfrom the group consisting of Terbogrel, Ridogrel, Ramatroban,Seratrodast, Ozagrel, Ifetroban, BM-531 and S18886.
 19. The method ofclaim 15, wherein the platelet ADP-receptor antagonist is selected fromthe group consisting of Plavix and clopidogrel.
 20. The method of claim15, wherein the platelet ADP-receptor antagonist is Plavix.
 21. A methodof qualifying a subject for treatment with a thromboxane A2 receptorantagonist, comprising: (a) perfusing a blood sample from said subjectthrough a channel in a perfusion device, wherein the channel has acoating which produces a blood deposit when exposed to blood and theperfusion device comprises a pump coupled to the outlet end of thehousing to draw the blood through the channel at a desired shear rate,producing a blood deposit, and wherein said blood sample is treated withan amount of a platelet ADP receptor antagonist sufficient to inhibitthrombosis at least approximately 20% relative to an untreated sample;and (b) wherein said blood sample is treated with an amount of aspirinsufficient to cause at least an approximately 50% inhibition ofthrombosis in a blood sample relative to an untreated sample; and (c)wherein a subject is qualified for treatment with a thromboxane A2receptor antagonist if less than approximately 50% inhibition ofthrombosis is observed in said blood sample relative to an untreatedsample.
 22. The method of claim 21, wherein the rate of blood deposit issufficient to determine the extent of aspirin responsiveness withinapproximately 5 minutes of the start of perfusion.
 23. The method ofclaim 21, wherein the thromboxane A2 receptor antagonist inducesdethrombosis.
 24. The method of claim 21, wherein thrombosis can beinhibited by increasing cAMP levels via P2Y₁₂ antagonism or via PGD2stimulation.
 25. The method of claim 1, wherein the perfusion deviceprovides real time measurements of (1) the thrombotic profile of aspirineffects on top of P2Y₁₂ antagonism in a collagen-coated perfusionchamber assay under arterial shear rates and (2) arachidonicacid/shear-induced platelet aggregation assay in whole blood under highshear rates.